Vector, element and method for inhibiting immune recognition

ABSTRACT

This invention relates to the use of Herpes Simplex Virus (HSV) immediate early protein ICP47, nucleic acid sequences coding for ICP47, and homologous proteins and nucleic acid sequences, to inhibit presentation of viral and cellular antigens associated with major histocompatibility class I (MHC class I) proteins to CD8+ T lymphocytes; this inhibition effectively increases infective persistence, which can, for example, improve the utility of viral gene therapy vectors. This invention also pertains to a method for the treatment of herpesvirus infections, wherein expression and/or activity of the ICP47 protein or its homologue is inhibited in order to increase immune recognition of herpesvirus-infected cells and other cells. This invention also pertains to a method for identifying drugs that interfere with the expression or function of ICP47 and its homologues, and which are useful in treating herpesvirus infections, and also pertains to the drugs so identified. Furthermore, this invention pertains to methods for the treatment and prevention of autoimmune diseases, tissue and organ transplant rejection, diabetes, multiple sclerosis, arthritis, and tissue damage accompanying ocular herpesvirus infections, wherein ICP47 or its homologue, or nucleic acids encoding such proteins, are introduced into the cells of a patient. In addition, this invention pertains to vector elements, vectors, polypeptides and polypeptide fragments that can be utilized for the forgoing purposes.

SUMMARY OF THE INVENTION

This invention relates generally to the use of isolated viral proteinsand isolated viral nucleic acids to inhibit the ability of the immunesystem to recognize and then destroy virus-infected cells or othercells. This invention also relates generally to the inhibition of viralgenes, MRNA and proteins in vivo in order to increase immune recognitionof infected cells and other cells. More specifically, the inventionrelates to the use of Herpes Simplex Virus (HSV) immediate early proteinICP47, DNA sequences coding for ICP47, and homologous proteins and DNAsequences, to inhibit presentation of viral and cellular antigensassociated with major histocompatibility class I (MHC class I) proteinsto T lymphocytes. This inhibition effectively increases infectivepersistence, which can, for example, improve the utility of viral genetherapy vectors. This invention also more specifically pertains tomethods for inhibiting expression and/or activity of the ICP47 protein,which can increase immune recognition of herpesvirus-infected cells andother cells, and which can, for example, serve as a means of treatingherpesvirus infections.

BACKGROUND OF THE INVENTION

The normal mammalian immune system responds to viral infection in avariety of ways. One important response is that T lymphocytes becomeable to recognize and kill virus-infected cells, while leavingnon-infected cells unharmed. Since viruses multiply by taking over thecell's machinery, when T lymphocytes kill the virus-infected cell theythereby limit the ability of the virus to reproduce itself.

The ability of T lymphocytes to kill only infected cells is mediated bythe ability of the infected cells to produce certain "signals". These"signals", which are protein-peptide complexes called majorhistocompatibility (MHC) complexes, are produced by mammalian cells inresponse to viral infection. These complexes are then transported to thesurface of the infected cells, where they are "displayed" to othercells, most notably T lymphocytes. As T lymphocytes circulate in thebody, they come into contact with cells that have MHC complexes on theirsurfaces. If those MHC complexes have associated with them viral orforeign antigens in the form of small fragments of viral or foreignproteins, receptors on the surfaces of the T lymphocytes becomeactivated, and the T lymphocytes are induced to kill those cells. Butwhen T lymphocytes come into contact with cells that do not have theviral or foreign antigens associated with the MHC complexes on theirsurface, the T lymphocytes do not disturb them. (Yewdell, J. W., andBennink, J. R., Cell biology of antigen processing and presentation tomajor histocompatibility complex class I molecule-restricted Tlymphocytes, Adv. Immunol. 52:1-123 (1992)).

There are two classes of MHC complexes, class I and class II. Theproduction and display of MHC class I complexes is fairly wellunderstood. Infected cells are able to degrade viral proteins to someextent, and short protein pieces, or peptides, are produced as a result.These peptides are transported from the nucleus or cytoplasm to theendoplasmic reticulum (ER) or to the Golgi apparatus; the ER and Golgiapparatus are convoluted, membranous intracellular organs involved inthe post-translational processing of proteins, and in their transport tothe cell surface. Once inside the ER or Golgi apparatus, the peptidesbind to the MHC class I protein α-chains and β-2-microglobulin, to forma trimolecular complex (Townsend, A., Ohlen, C., Bastin, J., Ljunggren,H. G., Foster, L., and Karre, K. Association of class I majorhistocompatibility heavy and light chains induced by viral peptides,Nature 340:443-448 (1989)). This complex is then transported to the cellsurface, where it can be recognized by T lymphocyte receptors. Receptorson the surface of a particular type of T lymphocytes, known asvirus-specific CD8+ T lymphocytes, specifically recognize the MHC classI complexes that are formed by the combination of MHC class I proteinsand peptides derived from a particular virus, and induce the CD8+lymphocytes to kill the cells that bear those complexes.

The presentation of MHC class I complexes and their recognition by CD8+T lymphocytes has been also implicated in a variety of human and animalafflictions other than viral infection. Perhaps the first roleidentified for MHC class I complexes was their role in tissue transplantrejection, which is why they are called "Major HistocompatibilityComplexes" (MHC). MHC Class I complexes appear to be of particularimportance in skin graft rejection. (Zijlstra, M., Auchincloss, H.,Loring, J., Chase, C., Russell, P., and Jaenisch, R., Skin craftrejection by β₂ -microglobulin-deficient mice, J. Exp. Med. 175:885-893(1992)). In addition, a large number of autoimmune diseases are believedto be the result of CD8+ T lymphocytes attacking cells displaying MHCclass I complexes. For example, there is evidence that attack by CD8+ Tlymphocytes plays a role in multiple sclerosis (see Steinman, L.,Autoimmune disease Sci. Amer. 269(3):106-114), diabetes (Oldstone, M. B.A., Nerenberg, M., Southern, P., Price, J., and Lewicki, H., Virusinfection triggers insulin-dependent diabetes mellitus in a transgenicmodel: role of anti-self (virus) immune response, Cell 65:319-331(1991)), and arthritis (Braun, W. E., HIA molecules in autoimmunediseases, Clin. Biochem. 25(3):187-191 (1992); Scarpa, R., Del Puente,A., di Girolamo, C., della Valle, G., Lubrano, E., and Oriente, P.,Interplay between environmental factors, articular involvement, andHLA-B7 in patients with psoriatic arthritis, Annals of Rheumatic Dis.51:78-79 (1992)).

Although viral infection usually results in the display and recognitionof MHC complexes, there are a number of animal viruses that are able topersist in the body, despite these mechanisms in the immune system thatusually detect and destroy infected cells. Some such persistent virusesproduce an extended or even constant infection, while others are able tobecome dormant or latent for long periods and then reappear to reinfectthe individual. It is now recognized that some of these viruses evadedetection by producing proteins that interfere with or block the cell'sability to make or display MHC class I complexes (Gooding, L. R., Virusproteins that counteract host defenses, Cell 71:5-7 (1992)).

Different persistent viruses appear to interfere with different stagesin the production and display of MHC complexes. For example, the E1agene of adenovirus type 12 produces a protein that blocks transcriptionof the MHC class I genes, thus preventing the production of the MHCclass I proteins themselves (Schrier, P. I., Bernards, R., Vaessen, R.T. M. J., Houweling, A., and van der Eb, A. J., Expression of class Imajor histocompatability antigens switched off by highly oncogenicadenovirus 12 in transformed rat cells, Nature 305:771-775 (1983)). TheE3 gene of human adenovirus types 2 and 5 produces a 19 thousand dalton(KD) protein that binds to the MHC class I proteins and causes them toremain sequestered or "stuck" in the ER or Golgi apparatus (Burgert, H.-G., and Kvist, S., An adenovirus type 2 glycoprotein blocks cellsurface expression of human histocompatibility class I antigens, Cell41:987-997 (1985)). Similarly, murine cytomegalovirus produces a proteinthat inhibits the transport of the completed protein-peptide complexesfrom the Golgi apparatus to the cell surface (del Val, M., Hengel, H.,Hacker, H., Hartlaub, U., Ruppert, T., Lucin, P., and Koszinowski, U.H., Cytomegalovirus prevents antigen presentation by blocking thetransport of peptide-loaded major histocompatibility complex class Imolecules into the media-golgi compartment, J. Exp. Med. 176:729-738(1992)). Using an apparently very different mechanism, myxoma virusappears to cause the MHC class I proteins to be removed from the cellsurface (Boshkov, L. K., Macen, J. L., and McFadden, G., Virus-inducedloss of class I MHC antigens from the surface of cells infected withmyxoma virus and malignant rabbit fibroma virus, J. Immunol. 148:881-887(1992)).

Herpes simplex virus (HSV) types 1 and 2 are persistent viruses thatcommonly infect humans; they cause a variety of troubling humandiseases. HSV type 1 causes oral "fever blisters" (recurrent herpeslabialis), and HSV type 2 causes genital herpes, which has become amajor venereal disease in many parts of the world. No fully satisfactorytreatment for genital herpes currently exists. In addition, although itis uncommon, HSV can also cause encephalitis, a life-threateninginfection of the brain. (The Merck Manual, Holvey, Ed., 1972; Whitley,Herpes Simplex Viruses, In: Virology, 2nd Ed., Raven Press (1990)).

A most serious HSV-caused disorder is dendritic keratitis, an eyeinfection that produces a branched lesion of the cornea, which can inturn lead to permanent scarring and loss of vision. Ocular infectionswith HSV are a major cause of blindness in North America. Immuneresponses play a major role in causing the tissue damage that resultsfrom recurrent ocular HSV infections, and T lymphocyte-mediatedresponses are a prominent cause of this damage. There is evidence thatthe CD8+ T cell subset is very important in these destructive immuneresponses (Hendricks, R. L., and Tumpey, M., Contribution of virus andimmune factors to herpes simplex virus type I-induced corneal pathology,Invest. Opthalmol. Vis. Sci. 31:1929-1939 (1990)).

On initial infection, HSV usually produces a generalized, acuteinfection, which is cleared by the body's normal immune response.However, during the acute phase, some virus particles invade sensorynerve cells, and there they are able to become latent, and survive longafter the acute infection has been cleared by the immune system, eventhough antibodies against them are abundant in the blood. They thenlater become reactivated and produce local infections. These are, asmight be expected, fairly rapidly cleared by the already-prepared immunesystem. (Zweerink, H. J., and Stanton, L. W., Immune response to herpessimplex virus infections: virus-specific antibodies in sera frompatients with recurrent facial infections, Infect. Immun. 31:624-630(1981)). This cycle is quite familiar to those who are prone to "feverblisters", which appear to be caused by sunlight-induced activation oflatent HSV particles in the lips.

Like certain other persistent viruses, it appears that HSV inhibitsimmune recognition of infected cells by interfering with the synthesis,transport or display of MHC class I complexes. One reason that this wasnot immediately appreciated by immunologists studying anti-HSV immunityis that in mouse models of HSV infection, the infected cells areprimarily killed by HSV-specific CD8+ T lymphocytes, which specificallyrecognize MHC class I protein-HSV peptide complexes; this suggests thatin these models, CD8+ T lymphocyte recognition is not stronglyinhibited. However, in humans, the HSV-infected cells are more oftenspecifically killed by HSV-specific T lymphocytes of another class,called CD4+, which recognize complexes composed of HSV-derived peptidesand MHC class II proteins. (Schmid, D. S. and Rouse, B. T., The role ofT cell immunity in control of herpes simplex virus, In: Herpes SimplexVirus: Pathogenesis, Immunobiology, and Control, B. T. Rouse, ed.(Berlin:Springer-Verlag) pp. 57-74 (1992)). Furthermore, it has beenfound that human fibroblasts that are infected with HSV are notrecognized and killed by HSV-specific CD8+ lymphocytes, but are killedby non-specific natural killer (NK) cells, which are not dependent onMHC class I complexes for recognition (Posavad, C. M. and Rosenthal, K.L., Herpes simplex virus-infected human fibroblasts are resistant to andinhibit cytotoxic T-lymphocyte activity, J. Virol. 66:6264-6272 (1992)).These findings suggest that recognition by CD8+ T lymphocytes isinhibited in human HSV infections.

Exactly what mechanism, what genes and what proteins might be involvedin HSV's ability to suppress immune recognition has, until discovery ofthe present invention, remained unknown. HSV resistance to T lymphocyterecognition was known to occur within 2 to 3 hours of infection, id.,but MHC class I expression on the surface of HSV-infected cells was notobserved to be markedly reduced until 14-20 hours after infection(Carter, V. C., Jennings, S. R., Rice, P. L. and Tevethia, S. S.,Mapping of a herpes simplex virus type 2-encoded function that affectsthe susceptibility of herpes simplex virus-infected target cells tolysis by herpes simplex virus-specific cytotoxic T lymphocytes, J.Virol. 49:766-771 (1984)). Furthermore, other cell-to-cell propagatedinactivation mechanisms have also been observed (York, I., and Johnson,D. C., Direct contact with herpes simplex virus-infected cells resultsin inhibition of lymphokine-activated killer cells due to cell to cellspread of virus, J. Infect. Dis. 168:1127-1132 (1993)).

The genome of herpes simplex virus type 1 is encoded on a linear,double-stranded DNA of about 152 kilobases. The HSV-1 genome has beencompletely sequenced. See: McGeoch, D., M. A. Dalrymple, A. J. Davison,A. Dolan, M. C. Frame, D. McNab, L. J. Perry, J. E. Scott and P. Taylor,The Complete DNA sequence of the Long Unique Region in the Genome ofHerpes Simplex Virus Type 1, J. Gen. Virol. 69: 1531-1574 (1988). Thegenome codes for about 76 proteins, many of which have been namedaccording to when in the infectious cycle they are produced. The proteinsequences for all of the HSV-1 proteins are known, having been deducedfrom their corresponding gene sequences. Furthermore, many years ofresearch has resulted in the identification of the function for many ofthese proteins. Nevertheless, there are still a number of proteinsencoded by the HSV-1 genome that have no known function.

One of the proteins whose function has remained unknown is theimmediate-early protein ICP47. Various researchers have given thisprotein other names, including IE12, Vmw12, and IE5. The gene for thisprotein is known as US 12, and is also known as α47. The coding regionof the US 12 gene is 264 base pairs long, which means that the ICP47protein is 88 amino acids long. Although ICP47 is observed to migrate ingel electrophoresis as a protein of about 12,000 daltons, the molecularweight, as calculated from its amino acid sequence, is 9792 daltons(McGeoch, D. J., Dolan, A., Donald, S., and Rixon, F. J., Sequencedetermination and genetic content of the short unique region in thegenome of herpes simplex virus type 1, J. Mol. Biol. 181:1-13 (1985)).

Various researchers have previously attempted to discern the function ofICP47, but prior to the present invention, without success. Deletion ofthe US 12 gene has been found to have no effect on infectivity(Mavromara-Nazos, P., Ackermann, M., and Roizman, B., Construction andproperties of a viable herpes simplex virus 1 recombinant lacking codingsequences of the α47 gene, J. Virol. 60:807-812 (1986)), and the mostrecent reported effort to determine the function of ICP47 concluded thatthe US 12 gene plays "no important role in the establishment and/orreactivation from latency" (Nishiyama, Y., Kurachi, R., Daikoku, T., andUmene, K., The US9, 10 11 and 12 genes of herpes simplex virus type 1are of no importance for its neurovirulence and latency in mice,Virology 194:419-423 (1993)).

Herpes Simplex Virus type 1 is but one member of an extended family ofviruses. HSV type 2 is a close relative; its genome is "collinear" withthat of HSV type 1, with "reasonable, but not identical, matching ofbase pairs". (Whitley, Supra at 1845). Other members of the humanherpesvirus family include cytomegalovirus, varicella-zoster virus,herpes virus 6, herpes virus 7, and Epstein-Barr virus. There are alsomore than 50 herpesviruses that infect more than 30 other animal species(Id.), including some that infect humans.

Herpes Simplex Virus Type 2 has a gene that corresponds to the HSV Type1 US 12 gene. It maps in the same genomic location, and produces aprotein that migrates as a 12,300 dalton protein on gel electrophoresis,which is similar to the migration of ICP47 (Marsden, H. S., Lang, J.,Davison, A. J., Hope, R. G., and MacDonald, D. M., Genomic location andlack of phosphorylation of the HSV immediate-early polypeptide IE 12, J.Gen. Virol. 62:17-27 (1982)). We have compared these gene sequences, andhave determined that the Herpes Simplex Virus types 1 and 2 ICP47proteins are 45% identical at the amino acid level, and 60% homologouswhen one allows substitution of similar amino acids.

Varicella-Zoster Virus does not appear to have a gene corresponding toUS 12 (Davison, A. J., and D. J. McGeoch, Evolutionary Comparisons ofthe S Segments in the Genomes of Herpes Simplex Virus Type 1 andVaricella-Zoster Virus, J. Gen. Virol. 67:597-611 (1986)), and thepseudorabies virus does not appear to contain a sequence correspondingto US 12 in the region encoding genes corresponding to other "uniquestretch" (US) genes (Zhang, G., and D. P. Leader, The Structure of thePseudorabies Virus Genome at the End of the Inverted Repeat SequencesProximal to the Junction with the Short Unique Region, J. Gen. Virol.71:2433-2441 (1990)). However, it is unclear whether the many otherherpesviruses contain such genes.

ADVANTAGES AND SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method forintroducing into cells an isolated gene or other protein coding nucleicacid sequence, the expression of which will at least partially interferewith one or more mechanism involved in specific recognition by Tlymphocytes.

It is also an object of the present invention to provide a method forintroducing into a virus an isolated gene or other protein codingnucleic acid sequence, the expression of which will reduce immuneresponses to the virus so that immune suppression or destruction ofvirus infected cells is reduced or delayed.

It is also an object of the present invention to provide a method forintroducing into cells a protein that will at least partially interferewith one or more mechanisms involved in specific recognition by Tlymphocytes.

It is a further object of the present invention to provide a method ofintroducing into virus-infected cells a protein that will enable thevirus to persist by at least partially avoiding recognition by Tlymphocytes.

It is another object of the present invention to provide a new elementfor a gene therapy vector, and to provide an improved gene therapyvector.

It is also an object of the present invention to provide a method forthe treatment of herpesvirus infections and to provide a method for theelimination of latent herpesviruses.

It is also an object of the present invention to provide a method foridentifying drugs useful in the treatment of herpesvirus infections, andto provide drugs identified thereby.

An additional object of the present invention is to provide a method tosuppress T lymphocyte-mediated organ or tissue transplant rejection.

Yet another object of the present invention is to provide a method forthe treatment of T lymphocyte mediated autoimmune diseases.

Still another object of the present invention is to provide a method forthe treatment of diabetes.

Another object of the present invention is to provide a method for thetreatment of multiple sclerosis.

Yet another object of the present invention is to provide a method forthe treatment of arthritis.

Another object of the invention is to provide a method for theprevention of tissue damage that occurs as a result of immune responsesto ocular herpes infections.

According to an embodiment of the invention, a method for improving theinfective persistence of a virus is described. This method comprisesintroducing into the viral genome an isolated nucleotide sequenceencoding a protein selected from the group of ICP47 of HSV type 1, IE 12of HSV type 2, proteins that are more than 40% homologous to ICP47 ofHSV-1, and fragments of any of the foregoing that are able to improvesaid infective persistence.

According to another embodiment of the invention, a vector element ableto suppress cell recognition by cytotoxic T lymphocytes byvector-infected cells is realized that comprises an isolated nucleotidesequence encoding a protein selected from the group of ICP47 of HSV type1, IE 12 of HSV type 2, proteins that are more than 40% homologous toICP47 of HSV-1, and fragments of any of the foregoing that are able tosuppress said recognition.

According to yet another embodiment of the invention, a vector elementable to suppress cell recognition by cytotoxic T lymphocytes byvector-infected cells is realized, which comprises an isolatednucleotide sequence selected from the group of the US 12 gene from HSVType 1, the HSV Type 2 gene encoding the IE 12 protein, nucleic acidsequences that are more than 40% homologous to the Herpes Simplex VirusType 1 US 12 gene, and fragments of any of the foregoing that are ableto supress said recognition.

According to yet another embodiment of the invention, a vector elementis realized that comprises the 714bp NruI - XhoI fragment of pRHP6,including part of the first exon, the intron, and the entire codingsequences of ICP47.

According to another embodiment of the invention, an adenovirus vectoris realized, comprising an adenovirus having the identifyingcharacteristics of AdICP47-1.

According to yet another embodiment of the invention, a method forinhibiting cell recognition by cytotoxic T lymphocytes is described,comprising introducing into cells an isolated nucleotide sequenceencoding a protein selected from the group of ICP47 of HSV type 1, IE 12of HSV type 2, proteins that are more than 40% homologous to ICP47 ofHSV-1, and fragments of any of the foregoing that are able to inhibitsaid recognition.

According to another embodiment of the invention, a method forinhibiting cell recognition by cytotoxic T lymphocytes is describedwhich comprises introducing into infected cells an isolated proteinselected from the group of ICP47 of HSV type 1, IE 12 of HSV type 2,proteins that are more than 40% homologous to the ICP47 protein ofHSV-1, and fragments of any of the foregoing that are able to inhibitsaid recognition.

According to yet another embodiment of the invention, a method for thetreatment of herpesvirus infections is provided, which comprises theintroduction into infected cells of a nucleotide sequence that iscomplementary to the MRNA sequence encoding a protein selected from thegroup of ICP47 of HSV type 1, IE 12 of HSV type 2, proteins that aremore than 40% homologous to ICP47 of HSV-1, and biologically activefragments of any of the foregoing, wherein the complimentary portion ofsaid nucleotide sequence is of sufficient length to inhibit thetranslation of said mRNA and thereby inhibit the production of saidprotein.

According to still another embodiment of the invention, a method for thetreatment of herpesvirus infections is provided, which comprises theintroduction into infected cells of an antibody specific for a proteinselected from the group of ICP47 of HSV type 1, IE 12 of HSV type 2,proteins that are more than 40% homologous to ICP47 of HSV-1, andantigenic fragments of any of the foregoing.

According to yet another embodiment of the present invention, a methodfor identifying drugs useful in treating herpesvirus infections isprovided, which comprises establishing a model cell system thatexpresses a protein that is selected from the group of ICP47 of HSV typeI, IE 12 of HSV type 2, proteins more than 40% homologous to ICP47, andfragments of any of the foregoing that exhibit the functionalcharacteristics of ICP47; adding amounts of candidate compounds tosamples of said model cells; and testing said samples for a traitdifferent from that observed in samples to which no such compound hasbeen added, said trait being selected from the group of suppressedsynthesis of the ICP47 homologue, decreased MHC class I proteinprocessing, and increased CTL lysis.

According to a further embodiment of the invention, a method is providedfor the prevention and treatment of autoimmune diseases, which comprisesintroducing into a patient's cells a biomolecule selected from the groupof an isolated nucleotide sequence encoding ICP47 of HSV type 1, anisolated nucleotide sequence encoding IE 12 of HSV type 2, isolatednucleotide sequences encoding proteins that are more than 40% homologousto ICP47 of HSV-1, the protein ICP47, the protein IE 12, proteins thatare more than 40% homologous to ICP47, and therapeutically effectivefragments of any of the foregoing.

Another embodiment of the invention provides a method for the preventionand treatment of tissue and organ transplant rejection, comprisingintroducing into the cells of said tissue or organ a biomoleculeselected from the group of an isolated nucleotide sequence encodingICP47 of HSV type 1, an isolated nucleotide sequence encoding IE 12 ofHSV type 2, isolated nucleotide sequences encoding proteins that aremore than 40% homologous to ICP47 of HSV-1, the protein ICP47, theprotein IE 12, proteins that are more than 40% homologous to ICP47, andtherapeutically effective fragments of any of the foregoing.

Still another embodiment of the present invention is a method for theprevention and treatment of diabetes, which comprises introducing intothe cells of a patient a biomolecule selected from the group of anisolated nucleotide sequence encoding ICP47 of HSV type 1, an isolatednucleotide sequence encoding IE 12 of HSV type 2, isolated nucleotidesequences encoding proteins that are more than 40% homologous to ICP47of HSV-1, the protein ICP47, the protein IE 12, proteins that are morethan 40% homologous to ICP47, and therapeutically effective fragments ofany of the foregoing.

A further embodiment of the present invention is a method for theprevention and treatment of multiple sclerosis, comprising introducinginto the cells of a patient a biomolecule selected from the group of anisolated nucleotide sequence encoding ICP47 of HSV type 1, an isolatednucleotide sequence encoding IE 12 of HSV type 2, isolated nucleotidesequences encoding proteins that are more than 40% homologous to ICP47of HSV-1, the protein ICP47, the protein IE 12, proteins that are morethan 40% homologous to ICP47, and therapeutically effective fragments ofany of the foregoing.

An additional embodiment of the invention is a method for the preventionand treatment of arthritis comprising introducing into the cells of apatient a biomolecule selected from the group of an isolated nucleotidesequence encoding ICP47 of HSV type 1, an isolated nucleotide sequenceencoding IE 12 of HSV type 2, isolated nucleotide sequences encodingproteins that are more than 40% homologous to ICP47 of HSV-1, theprotein ICP47, the protein IE 12, proteins that are more than 40%homologous to ICP47, and therapeutically effective fragments of any ofthe foregoing.

Another embodiment of the present invention is a method for reducingimmune reactions in ocular herpesvirus infections, comprisingintroducing into the ocular tissues of a patient a biomolecule selectedfrom the group of an isolated nucleotide sequence encoding ICP47 of HSVtype 1, an isolated nucleotide sequence encoding IE 12 of HSV type 2,isolated nucleotide sequences encoding proteins that are more than 40%homologous to ICP47 of HSV-1, the protein ICP47, the protein IE 12,proteins that are more than 40% homologous to ICP47, and therapeuticallyeffective fragments of any of the foregoing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(A) is a diagram of the recombinant HSV-1 virus denoted F-US5MHC;

FIG. 1(B) is an autoradiogram of electrophoretically separated ³⁵ Scysteine-labeled proteins that were expressed by uninfected Daudi cells,Daudi cells infected with recombinant F-US5MHC, and Daudi cells infectedwith wild-type strain HSV-1(F), and which were purified by immuneprecipitation with rabbit antipeptide 8 (α-p8) antiserum and rabbitanti-β2-m antiserum (α-β2-m);

FIG. 2(A-C) shows results of HSV-1-specific cytotoxic T lymphocyte lysisassays on (A) mouse fibrosarcoma (MC57) cells, (B) murine SVBALB cells,or (C) normal human fibroblasts (gwfb), each having first been infectedwith wild type HSV-1 (F), control virus F-US5β, or F-US5MHC;

FIG. 3(A-C) shows autoradiograms of electrophoretically separatedproducts from pulse-chase experiments where (A) MHC class 1 α chainproteins, (B) HSV-1 or HSV-2 glycoprotein D (gD), and (C) thetransferrin receptor were immunoprecipitated using monoclonalantibodies, and samples either were or were not treated withendoglycosidase H digestion prior to electrophoresis;

FIG. 4(A and B) shows (A) electrophoretic and (B) quantitative resultsof pulse chase experiments in which MHC class I products of uninfectedcells and cells infected with an HSV-1 mutant lacking the virion hostshut-off gene were detected with two antibodies, W6/32, which recognizesonly properly folded MHC class I proteins, and HC10, which recognizesboth properly folded and misfolded ones;

FIG. 5 shows the autoradiograms obtained after electrophoresis ofimmunoprecipitated, pulsed and chased MHC class I a chain proteins, bothwith and without prior endoglycosidase H digestion, from cells that wereinfected with either HSV-2 strain 333 or a mutant lacking thevirion-host shut-off function, or were alternatively infected with thosesame viruses after they were transcriptionally inactivated byirradiation with ultraviolet light;

FIG. 6 shows the autoradiograms obtained after electrophoresis ofimmunoprecipitated, pulsed and chased MHC class I a chain proteins, bothwith and without endoglycosidase H digestion, from cells infected withwild-type (KOS) HSV-1, and with HSV-1 mutants each defective in theirability to express a single gene, either the virion host shut-off gene(vhs⁻), or one of the genes for the immediate early proteins ICP4, ICP6,ICP0, ICP22, ICP27, and ICP47;

FIG. 7(A) is a diagram of the recombinant adenovirus vector designatedAdICP47-1;

FIG. 7(B) shows the electrophoresis pattern obtained when anICP47-specific antibody was used to precipitate radiolabeled proteinsproduced by cells infected with wild-type HSV-1 or with adenovirusvector AdICP47-1;

FIG. 7(C) shows the autoradiograms obtained after electrophoresis ofimmunoprecipitated, pulsed and chased MHC class I a chain proteins, bothwith and without endoglycosidase H digestion, from human fibroblastcells infected with wild-type HSV-1(KOS), AdICP47-1, or AddlE1 (whichdoes not express ICP47);

FIG. 8 shows the results of human cytomegalovirus-specific cytotoxic Tlymphocyte lysis assays on human MR fibroblast cells that wereuninfected, infected with human cytomegalovirus (CMV), or were infectedwith AdICP47-1 or AddlE1 followed by infection by CMV, and a similarassay on human allogeneic DG fibroblast cells that were subsequentlyinfected by CMV.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It is important to an understanding of the present invention to notethat all technical and scientific terms used anywhere herein, unlessotherwise defined, are intended to have the same meaning as commonlyunderstood by one of ordinary skill in the art; that techniques employedherein are also those that are known to one of ordinary skill in theart, unless stated otherwise; and that publications mentioned herein areincorporated by reference.

It is also important to note that reference to particular DNA fragments,genes, cDNAs, mRNAs, complementary strands, protein expression productsand the like, or to some subset of such related materials (e.g.,reference to DNA, where other related materials are not specificallylisted) is not intended to be limiting, but should be read to includeall such related materials that one of ordinary skill in the art wouldrecognize as being of interest or value in the particular context inwhich that discussion is presented. It is often possible to produce orprocure a biomolecule that is structurally related to or derived from astated material, and to use that biomolecule in a different but knownprocedure to achieve the same goals as those to which the use of asuggested method, material or composition is directed. For example, itis often possible to use RNA instead of DNA to carry geneticinformation. It is also possible to use certain nucleic acid analoguesin such applications. All such substitutions and modifications areincluded within the scope of the present invention.

It should also be noted that references to antibodies include bothpolyclonal and monoclonal antibodies, and also include sequences ofnucleic acid that bind specifically to particular proteins of interest,which nucleic acids are referred to as "nucleic acid antibodies" (Gold,L., Nucleic Acid Ligands, PCT Application No. WO 91/19813, publishedDec. 26, 1991).

Reference to a degree of homology between nucleic acids or proteinsmeans the percentage of nucleic acid bases or amino acids that arelocated identically in the sequences being compared, as is commonlyunderstood by those of ordinary skill in the art, unless it is specifiedthat similar amino acids should be allowed. Where specified, allowingsimilar amino acids to be substituted means that hydrophobic amino acidsmay be substituted for one another, as may cationic amino acids besubstituted for one another, etc., etc. Furthermore, in either case,although degrees of homology may be stated specifically, e.g., 40%, theyare meant to include further levels of homology, e.g, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90% 95% and 100%. Any mention of nucleicacid sequences encoding ICP47 or homologous proteins is intended toinclude all possible sequences of nucleic acids that might encode suchproteins, and is not intended to be limited to the sequences derivedfrom biological sources. Where no specific degree of homology isspecified, the term "homologous" means having at least a 25% degree ofhomology.

In addition, when homologues of ICP47 are mentioned, it is not intendedthat this be limited to those homologues that occur in nature; as isknown to those skilled in the art, by using modern molecular biologicaltechniques of site-directed mutagenesis, vector expression and the like,it is possible to produce new polypeptides, and the nucleic acidsequences that encode them, that are homologous to ICP47 and its codingsequence to various levels and in tremendously varied specific ways. Itis intended that such constructs, as well as the homologues that occurin nature, be included within the scope of this invention.

It should furthermore be understood that the scope of the presentinvention is not limited to full-length sequences of each of the nucleicacid and amino acid sequences described. As is well understood by thoseskilled in the art, it is often possible to prepare subfragments ofthose sequences, and for those subfragments, even small ones, to retainsome or all of the biological activity of the full-length sequences.Such subfragments are included within the scope of this invention.

The terms "virus" and "vector" as used herein are not intended to bemutually exclusive; to the contrary, they overlap considerably. A vectoroften is properly termed a virus, as that term is commonly understood. Avector often is simply a virus that has had a genetic element added, andboth the term virus and the term vector would properly apply. However, avector may have a form other than that of a virus. In addition, it isnot intended that the term virus only mean replicating virus particles;the term is intended, for example, to include non-replicating virusparticles, portions of viruses, and bacterial plasmids.

The term "heavy chain" is equivalent to the term "MHC class I α-chains";H-2K^(b) is a specific heavy chain derived from a particular strain ofmouse.

Experimental Procedures

Cells and Viruses

Many of the cell types used were of a common type, and are commerciallyavailable. Some cells used were obtained commercially, and others wereobtained from various laboratories. The types of cells used, thenon-commercial sources from which they were obtained (if any), and somekey references in which they are described are listed below:

Vero (African Green Monkey kidney) cells;

R-970-5 human osteosarcoma cells (Rhim, J. S., Cho, H. Y., and Huebner,R. J., Non-producer human cells induced by murine sarcoma cells, Int. J.Cancer 15:23-29(1975)), were obtained from K. Huebner and C. Croce ofthe Wistar Institute, Philadelphia, Pa.;

MC57 cells, which are mouse fibrosarcoma cells of the H-2^(b) haplotype(Zinkernagel, R. M., Adler, B., and Holland, J. J. Cell-mediatedimmunity to vesicular stomatitis virus infections in mice, Exp. CellBiol. 46:53-70 (1978)) were obtained from M. Buchmeier of the ScrippsInstitute, La Jolla, Calif.;

B6/WT-3 cells, which are mouse cells of the H-2^(b) haplotype (Pretell,J., Greenfield, R. S., and Tevethia, S. S., Biology of simian virus 40(SV40) transplantation antigen (T Ag). V. In vitro demonstration of SV40T Ag in SV40 infected nonpermissive mouse cells by the lymphocytemediated cytotoxicity assay, Virology 97:32-41(1979)) were obtained fromS. Tevethia at the University of Pennsylvania, Hershey, Pa.;

SVBALB cells, which are mouse cells of the H-2K^(d) haplotype (Gooding,L. R., Specificities of killing by T lymphocytes generated againstsyngeneic SV40 transformants: studies employing recombinants within theH-2 complex, J. Immunol. 122:1002-1008 (1979)), were obtained from K.Rosenthal at McMaster University, Hamilton, Ontario, Canada;

Daudi cells, which are EBV-transformed human lymphoblastoid cells inwhich the β₂ m genes are not expressed, (Klein, E., Klein, G., Nadkarni,J. S., Nadkarni, J. J., Wigzell, H., and Clifford, P., Surface IgM-kappaspecificity on a Burkitt lymphoma cell in vivo and in derived celllines, Cancer Res. 28:1300-1310 (1968));

293 cells, (Graham, F. L., Smiley, J., Russell, W. C., and Nairn, R.,Characteristics of a human cell line transformed by DNA from humanadenovirus 5 DNA. J. Gen. Virol. 36:59-74 (1977));

Normal human fibroblasts denoted gwfb, derived from a skin biopsy (usedbetween passages 10-20), EBV-transformed lymphoblastoid cell lines,obtained from K. Rosenthal of McMaster University, Hamilton, Ontario,Canada;

The human CD8+ CTL clone, MR-16E6, which is specific for humancytomegalovirus (HCMV) phosphoprotein 65, was isolated and propagated aspreviously described (Riddell, S. R. and Greenberg, P. D., The use ofanti-CD3 monoclonal antibodies to clone and expand humanantigen-specific T cells, J. Immunol. Methods 128:189-201 (1990);Riddell, S. R., Watanabe, K. S., Goodrich, J. M., Li, C. R., Agha, M.E., and Greenberg, P. D., Restoration of viral immunity inimmunodeficient humans by the adoptive transfer of T cell clones,Science 257: 238-257 (1992)).

Each of the forgoing cell strains were passaged in alpha-minimalessential media (a-MEM) containing 5 to 10% fetal bovine serum (FBS),unless otherwise indicated.

Many of the viruses used were also of a common type, and arecommercially available. Some viruses used were obtained commercially,and others were obtained from various laboratories. The types of virusesused, the non-commercial sources from which they were obtained (if any),and some key references in which they are described are listed below:

HSV-1 strain F (Ejercito, P. M., Kieff, E. D., and Roizman, B.,Characterization of herpes simplex virus strains differing in theireffect on social behaviour of infected cells, J. Gen.Virol. 2:357-64(1968)) was obtained from P. G. Spear at Northwestern University;

HSV-1 strain KOS (Smith, K. O., Relationships between the envelope andthe infectivity of herpes simplex virus, Proc. Soc. Exp. Biol.Med.115:814-16 (1964)) was also obtained from P. G. Spear at NorthwesternUniversity;

HSV-2 strain 333 (Kit, S., Kit, M., Qavi, H., Trkula, D., and Otsuka,H., Nucleotide sequence of the herpes simplex virus type 2 (HSV-2)thymidine kinase gene and predicted amino acid sequence of the thymidinekinase polypeptide and its comparison with the HSV-1 thymidine kinasegene, Biochim. Biophys. Acta 741:158-170 (1983)) was also obtained fromP. G. Spear at Northwestern University;

The HSV-1 deletion mutant VhsB, lacking the vhs gene UL41 (Smibert, C.A., and Smiley, J. R., Differential regulation of endogenous andtransduced β-globin genes during infection of erythroid cells with aherpes simplex type 1 recombinant, J. Virol. 64:3882-94 (1990)) wasobtained from J.Smiley at McMaster University, Hamilton, Ontario,Canada;

The HSV-2 mutant lacking the vhs gene (Smibert and Smiley, unpublished)was also obtained from J.Smiley at McMaster University, Hamilton,Ontario, Canada;

The ICPO deletion mutant, dlx3.1, (Sacks, W. R., and Schaffer, P. A.,Deletion mutants in the gene encoding the herpes simplex virus type 1immediate-early protein ICP0 exhibit impaired growth in cell culture, J.Virol. 61:829-839 (1987)) was obtained from P. Schaffer at theDana-Farber Institute, Boston, Mass.;

The ICP22 deletion mutant, R325-βT⁺, (Sears, A. E., I. W. Halliburton,B. Meignier, S. Silver, and B. Roizman, Herpes simplex virus 1 mutantdeleted in the α22 gene: growth and gene expression in permissive andrestrictive cells and establishment of latency in mice. J. Virol.55:338-346 (1985)) was obtained from B. Roizman at the University ofChicago;

The ICP6 deletion mutant, ICP6Δ, (Goldstein, D. J., and Weller, S. K.,An ICP6::lacZ insertional mutagen is used to demonstrate that the UL52gene of herpes simplex virus type 1 is required for virus growth and DNAsynthesis, J. Virol. 62:2970-2977 (1988)) was supplied by S. Weller ofthe University of Connecticut, Farmington, Conn.;

The ICP47 mutant, N38, (Umene, K., Conversion of a fraction of theunique sequences to part of the inverted repeats in the S component ofthe herpes simplex virus type 1 genome, J. Gen. Virol. 67:1035-1048(1986)) was obtained from K. Umene at the Kyushu University, Fukuoka,Japan;

The HSV-1 ICP4- mutant, d120, was propagated on complementing E5 cells(DeLuca, N. A., McCarthy, A.M., and Schaffer, P. A., Isolation andcharacterization of deletion mutants of herpes simplex virus type 1 inthe gene encoding immediate-early regulatory protein ICP4, J. Virol.56:558-570 (1985)), and was obtained from P. Schaffer at the Dana-FarberInstitute, Boston, Mass.;

The HSV-1 ICP27 deletion mutant, 5dl1.2, was propagated on complementing3--3 cells (McCarthy, A. M., McMahan, L., and Schaffer, P. A., HerpesSimplex virus type 1 ICP47 deletion mutants exhibit altered patterns oftranscription and are DNA deficient, J. Virol. 63:18-27 (1989)) was alsoobtained from P. Schaffer at the Dana-Farber Institute, Boston, Mass.;

The HSV-1 gD⁻ mutant F-US6kan was grown on complementing VD60 cells(Ligas, M. W., and Johnson, D. C. A herpes simplex virus mutant in whichglycoprotein D sequences are replaced by β-galactosidase sequences bindsto but is unable to penetrate into cells, J. Virol. 62:1486-94 (1988)).

Unless otherwise specified, and all the foregoing viruses werepropagated and titered on Vero cells.

Plasmids, Viral DNA and Vectors

Many of the plasmids used were of a common type, and are commerciallyavailable. Some plasmids used were obtained commercially, and otherswere obtained from various laboratories. These plasmids, thenon-commercial sources from which they were obtained (if any), and somekey references in which they are described are listed below:

Plasmid pTK173, containing the HSV-1 thymidine kinase gene (Smiley, J.R., Swan, H., Pater, M. M., Pater, A., and Halpern, M. E., Positivecontrol of the herpes simplex virus thymidine kinase gene requiresupstream DNA sequences, J. Virol. 47:301-310 (1983)) was obtained fromJ.Smiley at McMaster University, Hamilton, Ontario, Canada;

Plasmid pD6p, containing a lacZ gene cassette under control of the HSV-1ICP6 promoter (Goldstein, D. J., and Weller, S. K., An ICP6::lacZinsertional mutagen is used to demonstrate that the UL52 gene of herpessimplex virus type 1 is required for virus growth and DNA synthesis, J.Virol. 62:2970-2977 (1988)) was obtained from S. Weller of theUniveristy of Connecticut, Farmington, Conn.;

Plasmid pcKb, containing an EcoRI fragment including the murine H-2K^(b)gene inserted into plasmid pUC19 (Schonrich, G., Kalinke, U., Momburg,F., Malissen, M., Schmitt-Verhulst, A. M., Mallissen, B., Hammerling, G.J., and Arnold, B., Down-regulation of T cell receptors on self-reactiveT cells as a novel mechanism for extrathymic tolerance induction, Cell65:293-304 (1991)) was obtained from W. Jefferies at the University ofBritish Columbia, Vancouver, British Columbia;

Plasmid pVcβ2, containing the "a" allelle of murine β2-microglobulingene under the control of the SV40 promoter (Daniel, F., Morello, D., LeBail, O., Chambon, P., Cayre, Y., and Kourilsky, P., Structure andexpression of the mouse β2-microglobulin gene isolated from somatic andnon-expressing teratocarcinoma cells, EMBO J. 2:1061-1065 (1983)), wasalso obtained from W. Jefferies at the University of British Columbia;

Plasmid pRHP6, containing ICP4 and ICP47 sequences from HSV-1(KOS)(Perrson, R. H., Bacchetti, S., and Smiley, J. R., Cells thatconstitutively express the herpes simplex virus immediate-early proteinICP4 allow efficient activation of viral delayed-early genes in trans,J. Virol. 54:414-421 (1985)), was obtained from S. Bacchetti at McMasterUniversity, Hamilton, Ontario, Canada;

Plasmid pS456, which contains a BamHI-MscI fragment derived from plasmidpSS17 (Johnson, D. C., Frame, M. C., Ligas, M. W., Cross, A. M., andStowe, N. D., Herpes simplex virus immunoglobulin G Fc receptor activitydepends on a complex of two viral glycoproteins, gE and gI, J. Virol.61:2208-2216 (1988));

HSV-1 gD- mutant F-US6KAN (Smiley, J. R., Fong, B., and Leung, W. -C.,Construction of a double jointed herpes simplex virus DNA molecule:inverted repeats are required for segment inversion and direct repeatspromote deletions, Virology 113:345-362 (1981)).

Still other plasmids, viral DNA and vectors used in the experimentsdescribed herein were produced by recombinant DNA techniques that arewell known in the art (Sambrook, J., Fritsch, E. F., and Maniatis, T.,Molecular Cloning, a Laboratory Manual, 2nd Ed., Cold Spring HarborPress, 1989), and were prepared as follows:

To construct the recombinant HSV-1 expressing murine MHC class Iproteins, the 2.2 kb BamHI - NruI fragment from pvcβ2 containing theβ2-microglobulin gene was subcloned adjacent to the thymidine kinasepromoter from pTK173 and the 1.2 kb SalI - PvuII fragment of pcKbcontaining the murine H-2K^(b) gene was placed under the control of theICP6 promoter from pD6p . These β2-microglobulin/TK and H-2K^(b) /ICP6genes were then inserted into a unique NruI site in the US5 gene ofpS456, producing pS5MHC. pS5gal was produced by insertion of the 4.7 kbBamHI fragment containing the ICP6::lacZ cassette from pD6p into theNruI site in pS456.

Infectious viral DNA was prepared from Vero cells infected with theHSV-1 gD- mutant F-US6KAN. Vero cells were co-transfected with F-US6KANDNA and either plasmid pS5MHC or pS5gal, producing the viralrecombinants F-US5MHC or F-US5β, respectively, as previously described(Johnson, D. C., and Feenstra, V., Identification of a novel herpessimplex vitus type 1-induced glycoprotein which complexes with gE andbinds immunoglobulin, J. Virol. 61:2208-2216 (1987)). Recombinantviruses were repeatedly plaque purified on Vero cells, in which parentalF-US6KAN cannot replicate.

To construct an adenovirus vector expressing ICP47, designatedAdICP47-1, the 714bp NruI - XhoI fragment of pRHP6, including part ofthe first exon, the intron, and the entire coding sequences of ICP47,was inserted into the EcoRV - SalI region of pCA4 (C. Addison and F. L.Graham, unpublished), which contains the left side of the adenovirustype 5 genome with a deletion spanning the E1 region, into which isinserted the human cytomegalovirus (HCMV) immediate early promoter, apolylinker, and the SV40 polyadenylation signal, so that the ICP47coding sequences were placed next to the HCMV promoter; this producedthe plasmid p47NXE1. p47NXE1 was co-transfected with plasmid PBHG10,which contains full-length adenovirus 5 sequences but without thepackaging signal at the leftward side of the Ad5 genome (A. Bett and F.L. Graham, unpublished) into 293 cells. Recombinant adenoviruses, inwhich the ICP47/HCMV promoter cassette was inserted in the E1 region andcontaining a deletion in the E3 region, were plaque purified on 293cells and viral DNA was examined by restriction enzyme analysis.

Similarly, the control adenovirus vector Add1E1 was constructed byrescuing the plasmid pCA4 with the plasmid pJM17 (McGregory, W. J.,Bautista, D. S., and Graham, F. L., A simple technique for the rescue ofearly region I mutations in infectious human adenovirus type 5, Virology163:614-617 (1988)). The resulting virus lacked the same E1 and E3sequences as AdICP47-1, but did not encode ICP47 or any other foreigngene.

UV-inactivation of HSV

HSV-2(333) or HSV-2 (333 vhs-) virus stocks were prepared by suspendingVero cells in PBS containing 1% FBS, sonicating the cells extensively,and centrifuging the material at 1000 × g for 10 minutes to removeinsoluble material. The viruses were then diluted in PBS containing 1%FBS to 2 to 3 ml, placed in a 60 mm dish and subjected to UV light (3joules/cm² /sec using a bacteriostatic fluorescent tube) for 2 minuteswith constant stirring while on ice. UV-inactivated viruses were shownto be unable to express any viral proteins by immunoprecipitation andWestern blotting; the 333 stock retained vhs activity.

Antibodies

Antibodies used, their sources, and key references describing them are:

A hybridoma expressing monoclonal antibodies Y3, which reacts withH-2K^(b) complexed with β₂ -microglobulin, (Jones, B., and Janeway, C.A., Jr., Cooperative interaction of B lymphocytes with antigen-specifichelper T lymphocytes is MHC restricted, Nature 292:547-549 (1981)) wereobtained from the American Type Culture Collection (ATCC), Bethesda, Md;

A hybridoma expressing monoclonal antibody W6/32 (Parham, P.,Barnstable, C. J., and Bodmer, W. F., Use of a monoclonal antibody(W6/32) in structural studies of HLA-A,B,C antigens, J. Immunol.123:342-349 (1979)), which reacts with HLA-A, B, or C complexed with β₂microglobulin, was also obtained from ATCC;

Rabbit antiserum raised against a peptide from exon 8 of H-2K^(b)(Smith, M. H., Parker, J. M. R., Hodges, R. S., and Barber, B. H., Thepreparation and characterization of anti-peptide heteroantiserarecognizing subregions of the intracytoplasmic domain of class I H-2antigens, Mol. Immunol. 23:1077-1092 (1986)), which reacts with H-2K^(b)either complexed with, or free of, β₂ -microglobulin, was provided by B.Barber of the University of Toronto, Toronto, Canada;

Monoclonal antibody HC10, which reacts with unfolded human HLA-B and Cα-chains, and certain HLA A types (Stam, N. J., Spits, H., and Ploegh,H. L., Monoclonal antibodies raised against denatured HLA-B locus heavychains permit biochemical characterization of certain HLA-C locusproducts, J. Immunol. 137:2299-2306 (1986)), was provided by H. Ploeghat the Massachusetts Institute of Technology (MIT) in Boston, Mass.;

A rabbit antiserum raised to human β2m, which cross-reacts with mouse β₂m, was obtained from Dakopatts of Copenhagen, Denmark;

A rabbit polyclonal antiserum directed against peptide corresponding tothe C-terminus of ICP47 (Palfreyman, J. W., MacLean, J. B., Messeder,E., and Sheppard, R. C. Successful use of oligopeptides as immunogens inthe preparation of antisera to immediate-early gene products of herpessimpex virus type 1, J. Gen. Virol. 65:865-874 (1984)) was obtained fromH. Marsden at the Institute for Virology, Glasgow;

LP2, a monoclonal antibody specific for HSV gD (Minson, A. C., Hodgman,T. C., Digard, P., Hancock, D. C., Bell, S. E., and Buckmaster, E. A.,An analysis of the biological properties of monoclonal antibodiesagainst glycoprotein D of herpes simplex virus and identification ofamino acid substitutions that confer resistance to neutralization, J.Gen. Virol. 67:1001-1013 (1986)) was a gift of A. C. Minson at CambridgeUniversity, Cambridge, England;

T56/14, a monoclonal antibody specific for the transferrin receptor, wasobtained from Oncogene Science, Uniondale, N.Y.

Radiolabeling of cells, immunoprecipitations, and endoglycosidase Hdigestions

Human fibroblasts or Daudi cells were metabolically labeled with ³⁵ S-methionine and ³⁵ S -cysteine as previously described (Johnson, D. C.,and Feenstra, V., Identification of a novel herpes simplex vitus type1-induced glycoprotein which complexes with gE and binds immunoglobulin,J. Virol. 61:2208-2216 (1987)). For pulse-chase experiments, 100 mmplates of fibroblasts were labeled for 20-30 minutes with 100 μCi/mleach of ³⁵ S-methionine and ³⁵ S-cysteine (Dupont, Dorval, Quebec) thencell extracts were made using 1% Nonidet P40, 0.5% sodium deoxycholate,50 mM Tris-HCl, ph 7.5, 100 mM NaCl (NP40/DOC buffer) containing 2 mg/mlbovine serum albumin (BSA), and 1 mM phenyl methylsulfonyl fluoride(pulse) or cells were washed and incubated in alpha-MEM containing 1%FBS for 90 minutes then cell extracts were made (chase).

Immunoprecipitations were carried out as described previously (Id.).Cell extracts were stored overnight at -700° C. and were then clarifiedby centrifugation at 87,000 × g for 1 hr, then were mixed with ascitesfluids or serum and incubated on ice for 1-1.5 hr. Extracts to beimmunoprecipitated with monoclonal antibody HC10 were first heated at70° C. for 1 hour to partially denature MHC class I molecules, and thenthe extracts were cooled on ice. This treatment increased the fractionof class I molecules precipitated with HC10. Protein A-Sepharose wasadded and incubated a further 1.5-2 hours with mixing. The protein Abeads were collected by centrifugation, washed 3-4 times with NP40/DOCbuffer, and proteins were eluted by adding one volume of 2 × loadingbuffer (4% SDS, 20% glycerol, 4% β-mercaptoethanol and bromophenol blue)to each volume of beads and heating the beads at 100° C. for 5-10minutes. The stability of the MHC class I protein complex was determinedby first labeling class I proteins in uninfected or HSV-1(Vhs-B)-infected fibroblasts using ³⁵ S-methionine and ³⁵ S-cysteine(100 uCi/ml) for 1 hour, then chasing the label for 30 minutes. Cellextracts were made using NP40/DOC buffer containing 5 mg/ml BSA and 120TIU/ml aprotinin, and these extracts were incubated for 1 hour or 18hours on ice and then immunoprecipitated using monoclonal antibodiesW6/32 or HC10.

Endoglycosidase H (endo H) digestions were performed with extracts fromcells that had been labeled using a pulse-chase protocol. MHC class I,gD or transferrin receptors were immunoprecipitated using theappropriate antibodies, and proteins were eluted by suspending thesamples in denaturing buffer (0.5% SDS, 1% β-mercaptoethanol) andboiling them for 10 minutes. Half of each eluted protein sample wastreated with 1000 U endo H (New England Biolabs, Mississauga, Ontario,Canada) in reaction buffer (50 mM sodium citrate) and half was incubatedin reaction buffer alone, both for 3 hours at 37° C. The eluted proteinswere then subjected to electrophoresis through 14% polyacrylamide gelsfor MHC class I proteins and through 8.5% polyacrylamide gels for gD andthe transferrin receptor. The gels were impregnated with Enlightening(New England Nuclear, Boston, Mass.) and exposed to X-ray film or readusing a phosphorImager (Molecular Dynamics, Sunnydale, Calif.).

Cytotoxic T Lymphocyte lysis assays

Cytotoxic T Lymphocyte (CTL) lysis assays involving mouse cells wereperformed essentially as previously described (Pfizenmaier, K., Jung,H., Starzinski-Powitz, A., Rollinghoff, M., and H. Wagner, The role of Tcells in anti-herpes simplex virus immunity. I. Induction ofantigen-specific cytotoxic T lymphocytes, J. Immunol. 119:939-944(1977)). Briefly, C57BL/6 (H-2K^(b)) or BALB/c (H-2K^(d)) mice wereinfected with 1×10⁶ PFU of virus in the hind footpad. After 5 days, themice were anaesthetized and killed, and popliteal lymph nodes wereremoved and crushed through stainless steel mesh. The lymphocytes werecultured for 3 days at 37° C. in RPMI 1640/10% FCS/5×10⁻⁵ mMβ-mercaptoethanol (CTL medium). HSV-infected target cells (1-2×10⁴ cellsin 200 ml αMEM) were added to each well of a 96-well plate, the cellswere labeled with ⁵¹ Cr (Hanke, T., Graham, F. L., Rosenthal, K. L., andJohnson, D. C., Identification of an immunodominant cytotoxicT-lymphocyte recognition site in glycoprotein B or herpes simplex virusby using recombinant adenovectors and synthetic peptides, J. Virol.65:1177-1186 (1991)), and CTLs were added at various effector:targetcell ratios to a total of 200 ml CTL medium, and were incubated for 4hours at 37° C.

Cytotoxic T lymphocyte lysis assays involving human cells were performedusing a human CD8+ CTL clone, MR-16E6, which is specific for humancytomegalovirus (HCMV) phosphoprotein 65. Human fibroblasts wereinfected with a recombinant adenovirus vector, AdICP47-1 or AddlE1, for36 hours, then were subsequently infected with HCMV for 12 hours. Theywere then labeled with ⁵¹ Cr, mixed with the CTL clone using variouseffector to target (E:T) ratios, and were incubated for 5 hours at 37°C.

The results of the CTL lysis assays were determined by removing andcounting 100 μl from each well to obtain experimental release (ER) of ⁵¹Cr. Maximum release (MR) was obtained by counting aliquots aftertreatment with 1 M HCl. In each case samples were counted in a gammaradiation counter. Total release (TR) was calculated using the equationTR=MR+0.5ER. Non-specific release (NR) was determined using cells towhich no effectors had been added. Specific release (SR) was calculatedusing the equation SR=(ER-NR)/(TR-NR).

Results and Conclusions

Evidence that HSV-induced inhibition of MHC Class I complex presentationis not caused by blocking synthesis of MHC proteins, and isspecies-specific but not MHC class I-specific.

Previous observations that human CD8+ CTL were not able to lyseHSV-infected human fibroblasts and other normal diploid cells, eg.keratinocytes (Posavad, C. M. and Rosenthal, K. L., 1992, Supra)suggested that these cells were not recognized by CTL. To examine thisfurther with well-characterized murine CD8⁺, HSV-specific CTL, weconstructed a recombinant HSV-1, F-US5MHC, which expresses murine MHC(H-2^(b)) class I molecules. In this construct, the murine H-2K^(b) genewas placed under control of the HSV-1 ICP6 promoter, and the murine β₂-microglobulin gene was coupled to the HSV-1 thymidine kinase (tk)promoter; both of these constructs were then inserted into the HSV-1 US5(gJ) gene, which is not required for virus replication. The structure ofthis clone is diagrammed in FIG. 1(A). We also constructed a controlvirus, F-US5β, where the HSV-1 US5 gene was interrupted with theICP6::lacZ cassette from pD6p (Goldstein and Weller, 1988, Supra).Expression of the H-2K^(b) and β₂ -microglobulin proteins was theninvestigated by infecting Daudi cells, which do not express β₂-microglobulin, with F-US5MHC, with wild type HSV-1 strain F, or byleaving the cells uninfected (UN). Three hours after infection the cellswere labeled with ³⁵ S-methionine and ³⁵ S cysteine for 2 hours, andthen cell extracts were made, and the H-2K^(b) α-chain wasimmunoprecipitated using a rabbit anti-peptide 8 (α-p8); or, the β₂-microglobulin protein was immunoprecipitated using a rabbit anti-β2-mantiserum (α-β2-m). The results are shown in FIG. 1(B); molecular massmarkers are shown on the right. Expression of both the H-2^(b) α-chainand murine β2-microglobulin was detected in F-US5MHC-infected humanDaudi cells, which do not normally express β2-microglobulin, but not inDaudi cells infected with wild-type HSV type 1 strain F (lanes marked"F"). Other experiments confirmed that the H-2K^(b) α-chain wasexpressed in human fibroblasts and a number of other human cell typesinfected with F-US5MHC, and that this heavy chain protein reacted withthe anti-H2K^(b) conformation-dependent monoclonal antibody Y3 (data notshown).

F-US5MHC should in theory render any cell susceptible to lysis bymurine, H-2^(b) -restricted CTL. FIG. 2 shows that when cytotoxic Tlymphocytes (CTL) derived from C57BL/6 mice (H-2^(b)) infected withHSV-1(F) were used in CTL assays using effector to target ratios of 40,20, or 10:1, mouse fibrosarcoma H-2^(b) (MC57) target cells infectedwith F-US5MHC were efficiently lysed (FIG. 2(A)), as were (murineH-2^(d)) SVBALB cells (FIG. 2(B)). However, uninfected (UI) MC57 cellswere not lysed (FIG. 2(A)), nor were uninfected SVBALB cells or SVBALBcells infected with wild type HSV-1(F) or infected with control virusF-US5β. In other experiments, rat cells infected with F-US5MHC were alsorendered susceptible to lysis by H-2^(b) -restricted CTL (data notshown). In contrast, normal human fibroblasts (gwfb) (FIG. 2(C)) and apanel of other human cells (data not shown) were not lysed byHSV-specific, H-2^(b) -restricted CTL after infection with F-US5MHC; norwere uninfected (UI) human normal fibroblasts or fibroblasts infectedwith F-US5β or F-US5MHC. Therefore, these human cells were notrecognized by mouse cytotoxic T lymphocytes even though they expressedmouse MHC class I molecules. Together these results suggest that theHSV-induced inhibition of presentation to CTL is not related toinhibition of MHC class I synthesis and may be species-specific, but isnot MHC class I-specific.

Evidence that MHC class I molecules in HSV-infected cells are retainedwithin the ER/cis Golgi compartment

To further study MHC class I molecules in HSV-infected human cells, weused a pulse-chase protocol to examine intracellular transport andprocessing of class I α-chain molecules. Normal human fibroblasts (gwfb)were left uninfected or were infected with HSV-1(KOS) or HSV-2(333) for3 hr, then labeled with ³⁵ S-methionine and ³⁵ S-cysteine for 30 minutesand lysed (pulse: P) or the label was chased for 90 minutes (chase: C)before lysis. The results are shown in FIG. 3. In FIG. 3(A), MHC class 1α chain proteins were immunoprecipitated using monoclonal antibody HC10;in FIG. 3(B), HSV-1 or HSV-2 glycoprotein D (gD) were immunoprecipitatedusing monoclonal antibody LP2; and in FIG. 3(C), the transferrinreceptor was immunoprecipitated using the monoclonal antibody T56/14.The proteins were eluted from protein A beads and digested with endo H(+) or mock digested (-) at 37° C. before electrophoresis andautoradiography. In FIG. 3(D), human fibroblasts were infected withHSV-1 (F) for 0, 2, 4, 6, or 8 hours, were then pulse labeled for 30minutes as in (A), and the label was chased for 90 minutes. MHC class Imolecules were immunoprecipitated with antibody W6/32 and samples wereeither treated (+) or not treated (-) with endo H. A molecular massmarker of 45 KDa is indicated on the right.

The results show that Class I molecules immunoprecipitated from infectedor uninfected cells were digested with endoglycosidase H (endo H), whichremoves high-mannose but not fully processed oligosaccharides, as ameasure of glycoprotein transit through the medial and trans Golgicompartments (Townsend et al, 1989. Supra). MHC class I α-chains fromuninfected cells became resistant to endo H after a 90 minutes chaseperiod, while class I proteins from cells infected with HSV-1 or HSV-2remained sensitive to endo H (FIG. 3A). The inhibition of MHC class Itransport and processing in HSV-infected cells was apparently a specificeffect rather than a general one, since HSV-1 glycoprotein D (gD) andthe transferrin receptor were efficiently processed to become endoH-resistant during the 90 minutes chase period (FIG. 3B,C). When cellswere infected with HSV-1 and examined at various times after infection,alterations in the processing of MHC class I was first observed 2 hoursafter infection with HSV-1 and the effect was complete by 4 hours (FIG.3D). As expected, mouse H-2K^(b) class I molecules expressed in humanfibroblasts infected with F-US5MHC also remained in an endo H sensitiveform, yet H-2K^(b) and several other mouse MHC class I proteinsexpressed in HSV-infected mouse fibroblasts and other mouse cells becameendo H resistant (data not shown). These results demonstrate that MHCclass I complexes are retained in the endoplasmic reticulum/cis Golgi ofhuman fibroblasts, but not mouse cells, soon after infection with eitherHSV-1 or HSV-2.

It is well established that processing of N-linked oligosaccharidesoccurs in the Golgi apparatus as glycoproteins are transported from theendoplasmic reticulum (ER) (site of synthesis and initial glycosylation)to the Golgi then to the cell surface. When glycoproteins do not becomeprocessed they do not reach the cell surface. Therefore, lack ofprocessing indicated by endo H sensitivity (endo H recognizes immature,high mannose N-linked oligosaccharides but not mature complex N-linkedoligosaccharides) is indicative of lack of transport to the cell surface(for review see: Kornfeld, R., and Kornfeld, S., Assembly ofasparagine-linked oligosaccharides, Ann. Rev. Biochem. 54:631-664.(1985)).

Evidence that MHC I in HSV-infected cells is unstable.

MHC class I polypeptides produced in RMA-S and T2 cells, lacking theputative peptide transporter proteins, were found to be misfolded andunstable (Townsend et al, 1989, Supra; Townsend, A., Elliott, T.,Cerundolo, V., Foster, L., Barber, B., and Tse, A, Assembly of MHC classI molecules analyzed in vitro, Cell 62:285-295 (1990)). In order toexamine the stability and folding of class I molecules in HSV-infectedhuman fibroblasts, we carried out pulse-chase experiments in which wedetected the MHC class I products with two antibodies, one whichrecognizes only properly folded MHC class I proteins, and another whichrecognizes both properly folded and misfolded ones.

More specifically, in these experiments human fibroblasts were leftuninfected or infected for 4 hours with HSV-1 VhsB, a mutant lacking thevirion host shutoff gene, then the cells were radiolabeled for 1 hourwith ³⁵ S -methionine and ³⁵ S -cysteine and the label was chased for 30minutes. Cell extracts were mixed with antibodies immediately (1 hr) orwere incubated for 18 hours on ice before being mixed with antibodies.MHC class I proteins were immunoprecipitated using monoclonal antibodyW6/32, which recognizes only properly folded class I heavy/light chaincomplexes (Parham et al, 1979, Supra) or HC10 which, under theconditions used, recognizes misfolded as well as folded MHC class Iα-chains (Stam et al, 1986, Supra), by first heating the cell extractsfor 1 hour at 70° C. to denature the protein molecules. Afterimmunoprecipitation, samples were subjected to electrophoresis on 14%polyacrylamide gels, as shown in FIG. 4(A); a molecular weight marker of45 KD is shown at the right of the gel. A densitometric quantitation ofprotein bands corresponding to the class I α-chains was also performed,as shown in FIG. 4(B). The densitometric values obtained with uninfectedand HSV-infected cell extracts incubated for 1 hours and precipitatedwith HC10 were set at 100.

As is apparent from FIG. 4, There was a modest inhibition of MHC class Iα-chain synthesis in HSV-infected cells, even though an HSV-1 mutantunable to express the virion host shut-off function, vhs, was used(Smibert and Smiley, 1990, Supra); perhaps this was because ofcompetition between cellular and viral transcription and translationfactors. Densitometric quantitation of the protein bandsimmunoprecipitated by HC10 showed that there was no appreciableproteolytic degradation of the α-chain in either infected or uninfectedcells during the 18 hours incubation at 4° C. However, only a fractionof the class I α-chains present in extracts from HSV-infected cells wererecognized by W6/32. In the example shown, approximately 42% of theclass I molecules precipitated by HC10 (total number of molecules) wererecognized by W6/32 after 1 hours and about 30% of these moleculesdissociated during 18 hours at 4° C. (FIG. 4). In contrast, class Imolecules from uninfected cells were efficiently recognized by W6/32 andwere stable, as less than 5% of the molecules dissociated during the 18hours incubation. Therefore, it appears that MHC class I complexesformed in HSV-infected cells were misfolded and considerably less stablethan those formed in uninfected cells.

In these experiments, β2-microglobulin levels were not dramaticallyaltered by HSV infection (data not shown), and furthermore, thisβ2-microglobulin was available for binding to class I heavy chainsbecause 45% of the heavy chain could be recognized by monoclonal W6/32,which recognizes only class I complexes containing β2-microglobulin.

It is known that folding of MHC class I proteins in the ER is dependentupon trimerization of MHC class I heavy or alpha chain,β2-microglobulin, and small peptides derived from cellular or viralproteins (reviewed in Yewdell and Bennick (1990), Supra). Townsend etal. (1990, Supra) and numerous others have shown that MHC class Imolecules fail to assemble, fold improperly and are not transported tothe cell surface in mutant cells if peptides are not available in theER. Later studies indicated that this was because these mutant cellslack TAP proteins which "pump" peptides into the ER. In these mutantcells, eg. RMA-S or 0.174, MHC class I proteins remain sensitive to endoH and are misfolded. Moreover, the observed misfolding and instabilityof the MHC class I complexes in HSV-infected fibroblasts is similar tothat observed in TAP transporter-negative cell lines, and indicates thatpeptides are not associated with these MHC class I complexes inHSV-infected cells.

It has been found here that MHC class I proteins synthesized inHSV-infected cells have the same attributes, e.g., the class I proteinsremain endo H sensitive and are misfolded, as indicated by their lack ofrecognition by a conformationally sensitive monoclonal antibody. Sincetheir lack of processing means that the class I proteins do not reachthe cell surface in HSV-infected cells, one predicts that the class Iproteins would be defective in presenting viral antigens to Tlymphocytes.

Evidence that the HSV-1 immediate-early gene product ICP47 is requiredfor ER retention of MHC I.

HSV expresses three classes of gene products: immediate early (IE),early (E), and late (L), where IE proteins are required for thesynthesis of E and L proteins (Honess, R. W. and Roizman, B., Regulationof herpesvirus macromolecular synthesis. I. Cascade regulation of thesynthesis of three groups of viral proteins, J. Virol. 14:8-19 (1974)).However, a group of viral gene products including the vhs protein(McLaughlin, J., Addison, C., Craigie, M. C., and Rixon, F. J.,Noninfectious L-particles supply functions which can facilitateinfection by HSV-1, Virology 190:682-688 (1992)) and the VP16transactivator of IE proteins (Batterson, W., and B. Roizman.,Characterization of the herpes simplex virion-associated factorresponsible for the induction of a genes, J. Virol. 46:371-377 (1983))are incorporated into the virus particle and delivered into host cellsupon virus entry. Since MHC class I proteins were retained in the ERwithin 2 hours following HSV-1 infection (FIG. 2), it appeared thateither a virion structural protein or an immediate-early gene productwas responsible for the retention of those proteins.

To determine whether a virion structural protein was involved in thiseffect, stocks of HSV-2 were subjected to UV-inactivation so that thevirus particles retained vhs activity but were transcriptionally silent.Human fibroblasts were left uninfected, infected with HSV-2 (333), orwith HSV-2 (333-vhs⁻), a mutant derived from 333 which does not expressthe vhs function, using 10 plaque forming units/ml (PFU/ml). Othermonolayers of fibroblasts were treated with gradient purified,UV-inactivated virus particles derived from HSV-2 strain 333 or 333-vhs⁻at levels corresponding to 200 PFU/cell, and were incubated for 2 hoursat 37° C. The cells were labeled using the pulse-chase protocoldescribed for the experiments shown in FIG. 3, except that the pulse wasfor 20 min; then MHC class I proteins were immunoprecipitated usingantibody W6/32, and class I proteins digested (+) or not digested (-)with endo H, as also described for the experiments shown in FIG. 3. Theproteins were then subjected to electrophoresis on 14% polyacrylamidegels and exposed to X-ray film.

The results are shown in FIG. 5. In cells treated with relatively largequantities of UV-inactivated HSV-2 particles lacking the vhs protein(333-vhs⁻), MHC class I proteins were processed in an identical fashionto that in uninfected cells; processing to endo H resistant forms afterthe chase period was not significantly inhibited. UV-inactivated virusparticles derived from a HSV-2 strain which retained a wild type vhsgene (333-vhs+) produced a marked decrease in the expression of class Iproteins under these conditions, demonstrating that the virus particlesretained vhs activity after UV-inactivation which had shut off hosttranscription.

Since placing relatively high concentrations of structural proteins inthe presence of cells was inadequate to inhibit the processing of MHCclass I proteins to their endo H resistant forms, it appears that HSV-2structural proteins and the vhs protein, which are part of the virusparticle, are not sufficient to cause ER retention of MHC class Iproteins, and that transcription of HSV genes is required.

In order to determine whether HSV IE proteins were capable of inhibitingprocessing of MHC class I proteins, a panel of HSV-1 mutants unable toexpress the 6 IE proteins was analyzed. Human fibroblasts were leftuninfected (UN) or were infected with wild-type HSV-1(KOS), an HSV-1lacking the virion host shutoff gene (KOS-vhs⁻); HSV-1 mutant dl120,which is unable to express ICP4; HSV-1 mutant ICP6Δ, which is unable toexpress ICP6; HSV-1 mutant R325-βTK+, which is unable to express ICP22;HSV-1 mutant 5dl1.2, which is unable to express ICP27; or HSV-1 mutantN38, which is unable to express ICP47. Cells were infected for 3 hr,were labeled with ³⁵ S-methionine and ³⁵ S-cysteine for 20 min, and werethen immediately lysed (P) or the label was chased for 90 minutes (C)before lysis. MHC class I α-chains were immunoprecipitated usingmonoclonal antibody HC10 and the proteins either were treated with endoH (+) or were not treated (-) before electrophoresis andautoradiography.

The results of these experiments are shown in FIG. 6. In cells infectedwith HSV-1 d120, a mutant unable to express IE protein ICP4, class Iproteins remained endo H sensitive. Since ICP4 is strictly required forexpression of both E and L proteins (Dixon, R. A. F., and Schaeffer, P.A., Fine-structure mapping and functional analysis oftemperature-sensitive mutants in the gene encoding the herpes simplexvirus type 1 immediate early protein VP175, J. Virol. 36:189-203 (1980);Watson, R. J. and Clements, J. B., A herpes simplex virus type 1function continuously required for early and late virus RNA synthesis,Nature 285:329-330 (1980)), this shows that expression of only the HSV-1IE proteins is sufficient to inhibit class I processing and transport.Analysis of MHC class I proteins produced by mutants unable to produceIE proteins ICP6, ICP22, ICP27 and 1CP0 (FIG. 6) shows that thesemutants were still able to inhibit class I processing, as the proteinsremained endo H sensitive. This indicates that these IE proteins are notessential to the inhibition of MHC class I protein processing.

In contrast, mutant N38, which is unable to express ICP47 (Nishiyama etal. (1993), Supra) , did not block class I processing; the chasedsamples showed little or no endo H sensitivity, indicating thatprocessing was completed or nearly completed by that time. We note thatmutant N38 lacks coding sequences for the US9, US10, and US11 genes inaddition to the lack of ICP47 coding sequences; however, the US9, US10and US11 genes, which are early and late genes, were not expressed incells infected with mutant d120, which lacks the ICP4 gene function thatis required for expression of early and late genes, and cells infectedwith this mutant still caused inhibition of MHC Class I processing (seeabove). Thus, it is unlikely that any of these genes are involved inthis effect. In contrast, these results indicate that IE protein ICP47is involved in inhibition of MHC class I protein processing andtransport to the cell surface.

A second HSV-1 mutant, R3631, which lacks the ICP47 and US11 genes(Mavromara-Nazos, (1986), Supra), also failed to inhibit class Iprocessing (not shown). ICP47 is an IE gene which does not require ICP4for its expression, and it has no known effect on virus replication.Expression of the other IE genes, as well as E and L proteins, in cellsinfected with this mutant was normal (data not shown). This indicatesthat ICP47 is required for the observed inhibition of class I processingand transport, and that no other IE proteins are necessary.

Since MHC class I molecules must be transported to the cell surface sothat cells can be recognized by CD8+ T lymphocytes, the observedinhibition of class I processing and transport caused by ICP47 would beexpected to cause cells containing ICP47 to be resistant to thesecytotoxic T lymphocytes.

As a final and conclusive proof that the gene for ICP47 alone issufficient to produce the inhibition of MHC class I protein processingand transport, and that it is also sufficient to produce the inhibitionof CTL lysis that is observed in HSV infections, the ICP47 gene has beencloned into an adenovirus vector. Studies on this vector havedemonstrated that a vector carrying the ICP47 gene is able to producethe ICP47 protein, cause inhibition of MHC class I protein processingand transport, and inhibit cell lysis by cytotoxic T lymphocytes.Because these experiments are of great value in illustrating many of theuses for the ICP47 gene and its homologues that are part of thisinvention, they have been omitted here, and have instead been shown anddescribed as part of Example 1 hereinbelow.

Improving the infective persistence of a mammalian virus by insertinggenes for ICP47 or ICP47 homologues.

The use of viruses to serve as vectors for "gene therapy" is a promisingtechnique, which may in the future be widely practiced. Simply stated, agene encoding a protein having therapeutically desired effects is clonedinto a viral expression vector, and that vector is then introduced tothe target organism. The virus infects the cells, and then produces theprotein sequence in vivo, where it has its desired therapeutic effect.(See, e.g., Zabner, J., Couture, L. A., Gregory, R. J., Graham, S. M.,Smith, A. E., and Welsh, M. J., Adenovirus-mediated gene transfertransiently corrects the chloride transport defect in nasal epithelia ofpatients with cystic fibrosis, Cell 75:207-216 (1993)). However, onelimitation of this use of viral vectors for gene therapy is that theinfected cells get recognized and killed by the T lymphocytes, whicheventually results in the loss of the virus and its beneficialtherapeutic effect. Indeed, the concern that immune responses directedto viral proteins or to foreign gene therapy proteins might cause lossof gene therapy vectors is a major concern in the field of gene therapy(Id.).

The present invention offers a method by which the infective persistenceof viral gene therapy vectors might be improved, thus enhancing thetherapeutic value of those vectors. This method entails adding to thevector a sequence that encodes the ICP47 protein, the IE 12 protein ofHSV-2, or another protein substantially homologous to ICP47. Once thesesequences are added to the vector, the expression of those sequencesshould effectively inhibit CD8+ T lymphocyte recognition of infectedcells, and should thereby enhance the longevity and virility of thebeneficial virus.

One embodiment of this aspect of the invention is shown in Example 1hereinbelow, wherein the ICP47 gene has been isolated and inserted intoan adenovirus vector. This vector is thereafter able to inhibit theprocessing and transport of MHC class I proteins, and to thereby inhibitcell lysis by cytotoxic T lymphocytes that are directed against a viruswith which the cells were co-infected. This demonstrates that such avector or vector element could be used to protect the cells in whichthey reside from CTL-mediated cell lysis, whether the desired genetherapy gene was carried on that vector or on another vector with whichthe recipient was to be co-infected. Since it is the ICP47 gene productthat causes the inhibition, these experiments suggest that deliveringthe ICP47 protein or a protein substantially homologous to it into thecells would have a similar effect. Such proteins could be produced usingknown molecular biology methodologies (See: Sambrook et al, 1989,Supra).

Of course, the foregoing should not be viewed as limited to gene therapyvectors, as this method could be used to improve persistence in anyvirus in which such persistence may be desired.

The present invention also includes the vector elements that might beused to carry out this method. It is possible to use only the proteincoding regions for such purpose, or to use the entire genes, includingintrons and other elements. The 714bp NruI - XhoI fragment of pRHP6,including part of the first exon, the intron, and the entire codingsequences of ICP47, is one such element. Adenovirus vector AdICP47-1 isa gene therapy vector that includes this element.

Inserting genes for ICP47 or ICP47 homologues into somatic genes orsecondary vectors to inhibit cell recognition

As an alternative to adding the sequences encoding ICP47 or a homologousprotein to the DNA of a virus, it is also possible to introduce such agene into the somatic DNA of infected or uninfected cells, by methodsthat are well known in the art (Sambrook et al. 1989, Supra).Alternatively, it is also possible to introduce such a sequence intoinfected or uninfected cells by use of a secondary virus, i.e., one thatis not the virus that is expected to produce the immune response. Forexample, it may be desirable to introduce such a secondary viruscarrying an ICP47 coding sequence prior to the introduction of a viralgene therapy vector. This possibility is well illustrated by Example 1hereinbelow; the gene for ICP47 is carried on an adenovirus vector, yetis demonstrably able to inhibit cell lysis by cytotoxic T lymphocytesspecific for cytomegalovirus, with which the cells were co-infected.Thus, it is clear to one of ordinary skill in the art that one vectorcarrying the gene for ICP47 or a homologous protein could be used toinfect a patient, and that a second vector, e.g., a virus carrying agene to be introduced by gene therapy, could be used to infect that sameindividual; the first vector would inhibit recognition by CTL directedagainst either vector.

Additionally, it may be quite useful to introduce a sequence that codesfor ICP47 or a homologous protein into the cells of individualssuffering from autoimmune diseases, either by introducing it into thesomatic DNA or into a viral gene therapy vector, or alternatively, tointroduce the protein ICP47 or its homologue into the patient's cells.By doing so, it should be possible to limit the display of MHC Class 1complexes, and thereby limit autoimmune responses and symptoms of thedisease. This approach should be useful in the treatment of a number ofdisorders believed to be in part mediated by cytotoxic T lymphocytes,such as tissue and organ transplant rejection, diabetes, multiplesclerosis and arthritis, whether or not they are wholly "autoimmunedisorders" as that term is generally used. By reducing the recognitionof cells of involved tissues by cytotoxic T lymphocytes, the symptoms ofthe disorder should be reduced. It should also be of value to use such atherapy with patients at high risk for developing such disorders, evenif the damaging symptoms have not yet appeared. Furthermore, thisapproach should also be of value in the treatment of ocular herpesvirusinfections, where significant tissue damage occurs as a result ofcytotoxic T lymphocyte activity; this approach should lead to areduction in recognition of cells by T lymphocytes, and thereby reducetissue damage. Similarly, it should be possible to use this method totreat transplant recipients in order to limit CD8+ T lymphocyte-mediatedtissue rejection.

In some of the foregoing examples, it may only be necessary to introducethe genetic or protein elements into certain cells or tissues. Forexample, in the case of diabetes, introducing them into only thepancreas should be sufficient, and in the case of tissue or organtransplants, to only introduce them into the tissues or organ beingtransplanted. However, it may be more therapeutically effective and moresimple to treat all of the patient's cells.

Each of the above objectives should be equally attainable by adding tothe cells the ICP47 protein itself, or a homologous protein, which couldbe easily produced by known recombinant DNA methodologies. (Sambrook etal., 1989, Supra).

Treating persistent herpesvirus infections by use of antisense strandstargeted to an ICP47 homologous MRNA or gene.

There is now a considerable art regarding the use of so-called"anti-sense" polynucleotide sequences or analogs to prevent theexpression of proteins in vivo (for review see: Neckers, L. andWhitesell, L., Antisense Technology: biological utility and practicalconsiderations, Am. J. Physiol. 265 (Lung Cell. Mol. Physiol. 9:) L1-L12(1993)). The basic theory is that if you add to a cell a large number ofstrands of a nucleotide sequence that is complementary to the messengerRNA that is transcribed to produce a particular protein, these"anti-sense" strands will hybridize to the mRNA and limit or prevent itstranscription. This method could be used here to limit or prevent theexpression of ICP47 and homologous proteins by herpesviruses; as aresult, suppression of immune recognition by CD8+ T lymphocytes would bereduced, and the organism's immune system could more rapidly andeffectively kill the infected cells. It is possible that this will evenmake it possible to eliminate the recurring symptoms of herpesvirusinfection; e.g., in HSV-1 infections, it might be possible to preventrecurring "fever blisters".

Treating herpesvirus infections with antibodies against an ICP47homologous protein

Persistent herpesvirus infections may also be advantageously treated byintroducing monoclonal or polyclonal antibodies to the ICP47-homologousprotein that the given virus produces; this will limit or prevent thesuppression of MHC Type I complex expression, and thus allow for moreeffective immune clearing. Methods for intracellular introduction ofantibodies have been described (See. e.g. Carlson, J. R., A new use forintracellular antibody expression: inactivation of humanimmunodeficiency virus tyoe 1, Proc. Natl. Acad. Sci. U.S.A.90:7427-7428 (1993)).

Treating herpesvirus infections with drugs which interrupt or inhibitICP47

ICP47 appears to block availability of cellular or viral peptides whichwould normally be presented on MHC class I molecules, causing the MHCclass I molecules to become misfolded and preventing their transport tothe cell surface. Whatever the mechanism of ICP47 action, however, it isreasonable to conclude that ICP47 achieves its effect by interactionwith one or more intracellular protein or other molecule. Furthermore,it is clear that inhibition of MHC class I protein display depends onsynthesis of ICP47, and it seems reasonable to conclude that itseffectiveness is to at least some extent dose-dependent.

With these things in mind, it seems clear that by blocking the synthesisof ICP47 or its homologues, or by interfering with the interactionbetween ICP47 or its homologues and cellular molecules using drugsspecifically developed for this purpose, one would expect that cellsinfected with herpesvirus would be more readily recognized byanti-herpesvirus CD8+ T lymphocytes, leading to better recognition ofherpesviruses by the immune system, with the beneficial results ofreduced infection, decreased latency, and reduced symptoms.

The invention as described herein includes a method to screen for suchdrugs, as well as the drugs so identified. Quite simply, one need onlycreate a system that produces ICP47 or a homologue of it, add amounts ofcandidate compounds to that system, and determine whether synthesis ofICP47 or the homologue is inhibited, whether inhibition of theprocessing of MHC class I proteins decreases, or whether cytotoxic Tlymphocyte lysis of cells increases when compared to that same system inthe absence of the added compound. Candidate compounds could include awide spectrum of small molecules from which so-called "ethicalpharmaceuticals" are often identified, and could also include a widevariety of other compounds, including large and small syntheticcompounds, as well as many naturally-occuring or man-made biomolecules,including polynucleotides and polypeptides.

One could easily use one or more of the methods described herein toaccomplish this. For example, one might establish a model system thatproduces ICP47, either because it carries the coding gene in its genome,on a vector, or in a virus with which it has become infected. One couldthen test for decreased ICP47 synthesis by using the ICP47 antibodydetection method used to produce the results shown in FIG. 7; test foran increase in endo H resistance of the MHC class I complexes by using apulse-chase protocol as was used to produce the results shown in FIG. 6;and/or test for increased CTL lysis using the assay method used toproduce the results shown in FIG. 2 or FIG. 8.

EXAMPLE 1

Construction and characterization of a recombinant adenovirus vectorcarrying the coding sequence for HSV-1 ICP47

To determine whether other HSV gene products were required for theinhibition of antigen processing and whether ICP47 was sufficient forthis effect, we constructed a recombinant adenovirus vector, AdICP47-1.As shown in FIG. 7(A), the HSV-1 ICP47 gene was placed under the controlof the HCMV immediate early promoter and was inserted into the El regionof adenovirus type 5 to produce AdICP47-1. AdICP47-1 is replicationdefective because it lacks Adenovirus El sequences as well as E3sequences.

Expression of ICP47 proteins

The expression of ICP47 was examined by infecting human fibroblasts withHSV-1(F) using 10 PFU/cell or AdICP47-1 (Ad47) using either 10 or 100PFU/cell, or leaving fibroblasts uninfected (UN). Cells were labeled for2 hr, HSV-infected cells were radiolabeled from 1 to 3, 2 to 4, or 3 to5 hours after infection, and AdICP47-1-infected cells were radiolabeledfrom 36 to 38 hours after infection using ³⁵ S-methionine and ³⁵S-cysteine (50 uCi/ml of each). ICP47 was immunoprecipitated using anICP47-specific antipeptide serum and subjected to electrophoresis using16% polyacrylamide gels. As shown in FIG. 7(B), proteins of the samemolecular weight as ICP47 were produced by AdICP47-1 (lanes are marked"Ad47" in the figure legend), and these reacted with ICP47-specificantibodies. It therefore appears that the adenovirus vector AdICP47-1 isable to produce the ICP47 protein in vivo.

MHC class I transport and processing is inhibited

In order to determine whether the ICP47 proteins produced upon infectionof cells with adenovirus vector AdICP47-1 were able to inhibitintracellular MHC class I protein processing, human fibroblasts wereleft uninfected or were infected with HSV-1(KOS), AdICP47-1, or AddlE1(which lacks E1 and E3 sequences but does not express ICP47). Four hoursafter infection with HSV-1 or 36 hours after infection with anadenovirus, the cells were radiolabeled with ³⁵ S-methionine and ³⁵S-cysteine for 30 minutes and immediately lysed (P) or the label chasedfor 90 minutes (C) before lysis. MHC class I α-chains wereimmunoprecipitated using monoclonal antibody HC10 and treated with endoH (+) or not treated (-) before electrophoresis and autoradiography. Theresults are shown in FIG. 7(C); a molecular weight marker of 45 KDa isshown at the right.

These results show that MHC class I proteins produced in humanfibroblasts infected with AdICP47-1 remained sensitive to Endo H, aswith HSV-1-infected human fibroblasts. Class I molecules from cellsinfected with control adenovirus AddlE1, lacking E1 and E3 sequences,became largely endo H resistant. These results show that the ICP47protein produced by the recombinant adenovirus vector AdICP47-1 is ableto inhibit MHC class I protein processing in vivo.

Similar results were obtained with another adenovirus vector, AdICP47-3,where ICP47 coding sequences were placed under control of the SV40 earlypromoter and inserted into the E3 region so that the E1 sequences wereintact and the adenovirus vector was capable of replicating in humancells (data not shown). Therefore, other HSV gene products are notrequired for this effect and expression of HSV ICP47 is sufficient toprevent MHC I transport and processing.

Lysis by specific cytotoxic T lymphocytes is inhibited

In order to determine if ICP47 produced by an adenovirus vector couldinhibit recognition by cytotoxic T lymphocytes, human MR fibroblastswere left uninfected (UN) or were infected with AdICP47-1 or AddlE1 for36 hours, and were then infected with human cytomegalovirus (CMV) for 12to 16 hours. An allogeneic fibroblast cell line, DG, was also infectedwith CMV. The fibroblasts were then loaded with the radiolabel ⁵¹ Cr andmixed with various ratios (effector:target cell ratios or E:T ratios) ofa human cytomegalovirus-specific cytotoxic T lymphocyte clone, MR-16E6.Release of ⁵¹ Cr was determined after 5 hours, and percent specificlysis of the fibroblasts was calculated.

The results are shown in FIG. 8. Uninfected fibroblasts (UN) were notlysed by the HCMV specific clone (I.E., no ⁵¹ Cr was released aftermixing with the cytotoxic T lymphocytes) but 40% to 50% of the ⁵¹ Cr wasreleased from HCMV-infected fibroblasts (CMV) indicating that there wasspecific lysis of these cells. Lysis of allogeneic DG fibroblasts(Allo+CMV), which do not share the same MHC class I molecules as thecytotoxic T lymphocytes, did not occur, indicating that the cytotoxic Tlymphocyte clone lysed only target cells which shared the same MHC classI molecules. Prior infection of the fibroblasts with AdICP47-1 causedthe lysis of fibroblasts subsequently infected with HCMV (AdICP47 +CMV)to be reduced to background levels. By contrast, prior infection ofcells with AddlE1 had no effect on the lysis of cells infected with HCMV(AdICP47+CMV). Therefore, expression of ICP47 blocked lysis of the cellsby a cytotoxic T lymphocyte clone.

It is understood that the examples and embodiments described herein arefor illustrative purposes only, and that various modifications orchanges in light thereof will be suggested to persons skilled in theart; these are to be included within the spirit and purview of thisapplication and scope of the appended claims.

We claim:
 1. A method for inhibiting cell recognition by cytotoxic Tlymphocytes, comprising introducing into a mammalian cell an isolatednucleotide sequence encoding a herpes simplex virus protein whichinhibits the ability of said cell to present antigens associated withMHC class I proteins to T lymphocytes.
 2. The method of claim 1 whereinthe herpes simplex virus protein which inhibits antigen presentation bysaid cell is selected from the group consisting of ICP47 of HSV-1 andIE12 of HSV-2.
 3. The method of claim 1 wherein the herpes simplex virusprotein which inhibits antigen presentation by said cell is ICP47 ofHSV-1.
 4. The method according to claim 1 wherein the nucleotidesequence is introduced through the use of a viral vector.
 5. The methodaccording to claim 1 wherein the nucleotide sequence is introducedthrough the use of a viral vector separate from and in addition to aexpression vector with which the cell is also infected.
 6. A recombinantexpression vector comprising a promoter operably linked to a nucleotidesequence encoding an HSV-1 ICP47 protein or an HSV-2 IE12 protein, whichexpression vector, when present in a mammalian cell, inhibits theability of said cell to present antigens associated with MHC class Iproteins to T lymphocytes, and so inhibits recognition of said cell bycytotoxic T lymphocytes.
 7. An expression vector according to claim 6wherein the nucleotide sequence encoding an HSV-1 ICP47 proteincomprises the 714 bp NruI-XhoI fragment of pRHP6, including part of thefirst exon, the intron, and the entire coding sequences of ICP47.
 8. Anexpression vector according to claim 6 which is a recombinant viralexpression vector.
 9. A vector according to claim 8 wherein said vectoris ADICP47-1, or wherein said vector has the activity of ADICP47-1 ofinhibiting the ability of a mammalian cell to present antigensassociated with MHC class I proteins to T lymphocytes, and so inhibitingrecognition of said cell by cytotoxic T lymphocytes, when present insaid cell.